The long term objective of this subproject is to decipher the role of calponin (CaP) in the regulation of smooth muscle contraction. The biochemical properties of CaP strongly suggest that it can regulate the contractility of smooth muscle by modifying the interaction between actin and myosin. Results of experiments using exogenously added CaP or CaP antagonist seem to support this notion. On the other hand, in situ localization studies show that Cap is primarily localized in the cytoskeletal rather than the actomyosin regions of chicken gizzard cells. These findings suggest several possibilities: 1) CaP is localized in the actomyosin regions in the relaxed state where it functions by inhibiting actin-myosin interaction; upon stimulation it migrates out of the actomyosin regions to the cytoskeletal regions. 2) CaP is not localized in the actomyosin regions regardless of contractile state and type of tissue; instead, it is localized in the cytoskeletal regions where it modulates contractibility via the cytoskeleton. 3) CaP is not involved in contractibility. In this process we will test these possibilities by carrying out the following experiments: 1) Determine the in situ localization of CaP as a function of contractile state and cell type. 2) Determine the in vivo stoichiometries of CaP and various contractile and cytoskeletal proteins. 3) Study the interaction between CaP and various smooth muscle proteins. 4) Determine the effects of CaP and CaP antagonists on the contractility and cytoskeletal structure of smooth muscle cells. Results derived from this project may reveal the true physiological function of CaP and uncover novel concepts in smooth muscle physiology such as the involvement of the cytoskeleton in contractility.